The homeodomain family of proteins play a fundamental role in the specification of body plan, pattern formation, and the determination of cell fate. In question was whether a particular protein, called prospero, was truly a homeodomain protein, given unclear inferences in the literature. Using a technique called threading, we assessed whether the non-canonical prospero sequence could form the three-helical homeodomain structural motif despite its low sequence similarity to the class. Through these experiments, we were able to assign definitively that the prospero proteins as being homeodomains, and in the process, were able to deduce which residues were responsible for maintaining the three-dimensional structure of these proteins.Upon completion of this work, our focus shifted to two genetic disorders caused by defects in Pitx2, another homeodomain protein. Mutations in the human Pitx2 gene in 4q25-q26 lead to two eye-related disorders, called Rieger Syndrome and iridogoniodysgenesis; sequelae include iris hypoplasia and the eventual development of glaucoma. Documented mutations leading to these disorders were subjected to threading analysis, showing that the mutations lead to the loss of critical pairwise interactions within the Pitx2 protein, meaning that the protein cannot adopt the structure required for proper function. In the past, biochemical observations explaining this disease were documented simply as loss-of-function; this is the first time that a concrete, structural underpinning for the development of these disorders has been proposed. Information on this and other work can be found at the Homeodomain Resource, at http://genome.nhgri.nih.gov/homeodomain/. - Protein structure, molecular modeling, development, protein folding, homeodomain